The gene for the neuronal apoptosis inhibitor protein (NAIP) has been identified in the q13 region of chromosome 5. Mutations in this gene have been diagnosed in individuals with Type I, II and III Spinal Muscular Atrophy. The amino acid sequence of the neuronal apoptosis inhibitor protein is provided and homology to viral apoptosis proteins demonstrated.
In order to facilitate reference to various journal articles in the discussion of various aspects of this invention, a complete listing of the reference is provided at the end of the disclosure. Otherwise the references are identified in the disclosure by first author""s name and publication year of the reference.
The childhood spinal muscular atrophies (SMAs) are a group of autosomal recessive, neurodegenerative disorders classified into three types based upon the age of onset and clinical progression (Dubowitz et al., 1978; Dubowitz et al., 1991). All three types are characterized by the degeneration of the alpha motor neurons of the spinal cord manifesting as weakness and wasting of the proximal voluntary muscles. Type I SMA is the most severe form with onset either in utero or within the first few months of life. Affected children are unable to sit unsupported and are prone to recurrent chest infections due to respiratory insufficiency, thus rarely surviving the first few years of life (Dubowitz et al., 1978; Dubowitz et al., 1991). This acute form, with a carrier frequency of {fraction (1/60)} to {fraction (1/80)}, is one of the most frequent fatal autosomal recessive disorders. Affected children with Type II SMA never walk unaided and although the prognosis is variable, such children may die in adolescence. Those affected with Type III SMA maintain independent ambulation but develop weakness any time between the age of 3 to 17 years manifesting a mildly progressive course (Dubowitz et al., 1978; Dubowitz et al., 1991).
In 1990, all three childhood forms of SMA were genetically mapped to the long arm of chromosome 5 at 5q11.2-13.3 (Brustowitcz et al., 1990; Gilliam el al., 1990; Melki et al., 1990). Subsequent multi-point linkage analyses and the identification of recombinant events have further localized the genetic defect to the region flanked centromerically by D5S435/D5S629 (Soares et al., 1993; Wirth et al., 1993, Clermont et al., 1994)) and telomerically by MAP1B/D5S112 (Wirth et al., 1994; MacKenzie et al., 1993; Lien et al., 1991). This interval has been refined by the more recent identification of recombination events indicating that the SMA gene lies distal to CMS-1 (Yaraghi et al., submitted to Human Genetics; van der Steege, et al., submitted to Human Genetics) and proximal to D5S557 (Francis et al., 1993). We and others have detected chromosome 5-specific repetitive sequences with particular abundance in the D5S629/CMS-D5S557 region (Francis et al., 1993; Thompson et al., 1993) which has impeded the isolation and ordering of both clones and simple tandem repeats. An array of cosmid clones spanning the 200 kb CMS-1 (Kleyn et al., 1993)/CATT-1 (Burghes et al., 1994, McLean et al., in press)/D5F150/D5F149/D5F153 (Melki et al., 1994) region within this interval has been constructed.
We established a contiguous array of YAC clones encompassing the SMA containing D5S435-D5S112 interval of 5q13.1. We then discovered a gene within this interval of 5q13.1 which coded for a neuronal apoptosis inhibitor protein (NAIP). Further studies demonstrated that a deletion in this gene was found in Type I, II and III Spinal Muscular Atrophy.
A gene encoding a neuronal apoptosis inhibitor protein (NAIP) was discovered in the q13 region of human chromosome. According to an aspect of the invention, the cDNA sequence coding of the neuronal apoptosis inhibitor protein is provided and set out in Table 4 (SEQ ID NO: 1). According to another aspect of the invention, the predicted amino acid sequence of the neuronal apoptosis inhibitor protein is provided from the cDNA sequence.
According to another aspect of the invention, a deletion of the neuronal apoptosis inhibitor protein gene was discovered in persons with Type I, II and III Spinal Muscular Atrophy disease. The discovery of the neuronal apoptosis inhibitor protein gene deletion provides a diagnostic indicator for use in the diagnosis of Spinal Muscular Atrophy.
In order to facilitate a further description of various aspects of the invention, reference will be made to various Figures of the drawings. A brief description of the drawings follows this invention summary section.
According to a further aspect of the invention, a human gene is provided which maps to the SMA containing region of chromosome 5q13. The gene comprises exons 1 through 17 of approximately 5.5 kb and having a restriction map for exons 2 through 11, as shown in FIG. 8.
According to a further aspect of the invention, exons 1 through 17 have a restriction map for exons 2 through 16, as shown in FIG. 9D.
According to another aspect of the invention, a human gene of the above aspects wherein exons 5 through 16 code for the NAIP protein having an amino acid sequence biologically functionally equivalent to the amino acid sequence of SEQ ID NO: 2.
According to another aspect of the invention, the human gene of the above aspects have exons 5 through 16 with a cDNA sequence biologically functionally equivalent to the cDNA sequence of SEQ ID NO: 1.
According to another aspect of the invention, a purified nucleotide sequence comprises genetic DNA, cDNA, mRNA, anti-sense DNA or homologous DNA corresponding to the cDNA sequence of SEQ ID NO: 1.
According to another aspect of the invention, a DNA molecule sequence coding for the NAIP protein having SEQ ID NO: 2.
According to another aspect of the invention, a purified DNA sequence consists essentially of DNA SEQ ID NO: 1.
According to another aspect of the invention, a purified DNA sequence consists essentially of a DNA sequence coding for amino acid SEQ ID NO: 2.
According to another aspect of the invention, a purified DNA sequence comprises at least 18 sequential base of SEQ ID NO: 1. DNA probes, PCR primers, DNA hybridization molecules and the like may be provided by using the purified DNA sequence of at least 18 sequential bases.
According to another aspect of the invention, use of the DNA sequences of the above aspects in the construction of a cloning vector or an expression vector.
According to another aspect of the invention, NAIP protein encoded by the above DNA sequences.
According to another aspect of the invention, NAIP protein comprising an amino acid sequence biologically equivalent to the amino acid sequence of SEQ ID NO: 2.
According to another aspect of the invention, NAIP protein consisting essentially of the amino acid sequence of SEQ ID NO: 2.
According to another aspect of the invention, NAIP protein fragment comprises at least 15 sequential amino acids of SEQ ID NO: 2.
According to another aspect of the invention, use of the above amino acid sequences in the production of hybridomas.
According to another aspect of the invention, a method is provided for analyzing a biological sample to determine the presence or absence of a gene encoding NAIP protein.
The method comprises:
i) providing a biological sample derived from the SMA containing region q13 of chromosome 5;
ii) conducting a biological assay to determine presence or absence in the biological sample of at least a member selected from the group consisting of:
a) NAIP DNA SEQ ID NO: 1, and
b) NAIP protein SEQ ID NO: 2.